Treated tissue for implantation and methods of treatment and use

ABSTRACT

Tissue which is suitable for transplant is treated with a growth factor and cells which populate the tissue and native cells must be removed, they cannot be &#34;masked&#34; reduce immunogenicity; this increases the longevity of the tissue upon transplant. The preferred growth factor is basic fibroblast growth factor, and the preferred cells are fibroblasts. The tissue can be an allograft or xenograft taken from a cow, pig or other mammal.

RELATED U.S. PATENT APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 08/023,492, filed on Feb. 26, 1993 now U.S. Pat. No. 5,772,695which is a continuation of U.S. patent application Ser. No. 07/664,902filed on Mar. 5, 1991 now U.S. Pat. No. 5,192,312 entitled "TreatedTissue For Implantation and Methods of Treatment and Use".

During the last 20 years, allograft heart valve transplantation in theUnited States has increased from about 50 to 2,000 per annum. Because ofthe increase in demand, particularly in pediatric cases, utilization ofallograft heart valves is now limited by the supply of donated humanhearts.

The invention described herein relates to transplantable tissue, such asheart valves, which is treated to reduce potentially untoward reactionsto the tissue which would otherwise result upon transplant. Implantabletissue has in the past been taken from patients and reimplanted into thesame patient in a different site, such as with burn victims who requireskin grafts and coronary bypass patients who require coronary arterialreplacement using sections of saphenous veins.

Similarly, organs such as kidneys have been transplanted allogeneicallyfrom one sibling to another in an effort to minimize immunologicallymediated reactions by the transplant recipient, which would result inorgan rejection. These patients, as well as patients receivingtransplant organs from donors other than siblings, are frequentlyadministered drugs to suppress the immune system. While theimmunological response to transplant tissue may be suppressed throughthe use of immunosuppressant drugs to minimize tissue rejection,immunosuppressant therapy is general in nature. Hence, immunosuppressantdrugs also tend to suppress the immune response, which reduces thetransplant patient's ability to combat infection.

The supply and ready availability of transplantable organs and grafttissue has been far outdistanced by the demand for such tissue over thepast several years, and there is a long-felt need for an increase in thesupply of such organs and tissue. This need remains to an extentunfilled, even taking into account the various synthetic tissues andmechanical organs which are presently available.

Bioprosthetic grafts are typically superior to mechanical prostheticdevices for various reasons. For example, mechanical heart valves aretypically more prone to cause thromboembolism than bioprosthetic grafts.Moreover, mechanical equipment failures typically occur suddenly andwithout warning, resulting in emergency situations requiring surgicalintervention and replacement of the artificial prosthetic device.Bioprosthetic heart valve grafts do not typically fail suddenly when aproblem occurs. Rather, if there is a secondary valve failure, the valvetends to wear out gradually over time. This gives the patient andtreating physician some advance warning that a graft prosthesis failureis likely to occur.

The invention described herein relates to xenogeneic or allogeneictissues made suitable for transplant into a patient by replacing nativecells within the tissue with autogenous or allogeneic cells. Thesemodified grafts combine the advantages of bioprosthetic valves withimmunological tolerance on the part of the recipient and the ability tomaintain and repair the extracellular matrix.

There have been attempts at producing artificial tissues and organs inthe past with varying degrees of success.

Steinberger, U.S. Pat. No. 4,407,787, relates to a dressing comprised ofcollagen and a resorbable biopolymer. The dressing istissue-agglutinable, such that the dressing adheres to tissue and causeshemostasis.

Caplan, et al., U.S. Pat. No. 4,609,551, relates to a process forstimulating bone and cartilage growth, utilizing a soluble bone protein.The bone protein is combined with cells such as fibroblasts, and themixture may be injected into the site of a joint cavity articularsurface defect. Alternatively the bone protein and cells may beimplanted in a fibrin clot. The fibroblasts differentiate to formreplacement cartilage tissue.

Nevo, et al., U.S. Pat. No. 4,642,120, relates to a gel-type compositionfor repairing bone and cartilage defects. The gel contains mesenchymalcells which differenciate into cartilage cells through the influence ofchondrogenic inducing factor in combination with fibrinogen,antiprotease and thrombin.

Bell, U.S. Pat. No. 4,485,096, relates to a tissue equivalent fortreatment of burns or skin wounds and to fabricated prostheses. Ahydrated collagen lattice is contracted with a contractile agent, e.g.,fibroblasts or blood platelets, to create a collagen lattice which maythen be populated with keratinocytes, thus forming a skin equivalent.Alternatively, glandular cells, such as pancreatic beta cells, orhepatocytes can be grown on the collagen lattice to produce a pancreasor liver tissue "equivalent". Bone equivalents can also be formed fromthe contracted collagen matrix described above in combination withdemineralized bone powder.

Bell, U.S. Pat. No. 4,539,716, similarly relates to synthesizedequivalents for blood vessels and glandular tissue. A contractile agentis used to contract the collagen lattice axially around an inner core.Additional layers containing capillary beds, blood vessels and glandularstructures are then constructed.

Bell, U.S. Pat. No. 4,546,500, relates to the fabrication of bloodvessels and glandular tissues utilizing a collagen lattice contractedaxially around an inner core and combined with a plastic mesh sleeve.The plastic sleeve is sandwiched between layers of the matrix toreinforce the structure.

Bell, et al., U.S. Pat. No. 4,835,102, relates generally to tissueequivalent test systems, and includes tissue equivalents for epithelial,connective, cartilage, bone, blood, organs and glandular tissues as wellas blood vessels. The tissue equivalent is composed of cultured cellswhich are derived from the endogenous tissue and incorporated into acollagen lattice.

Bell, et al., PCT Application WO 86/02273 published Apr. 24, 1986,relates to methods of forming living tissue equivalents, which utilize acollagen matrix contracted to form a lattice in a nutrient medium. Theinitially acidic collagen system is precipitated by raising the pHsufficiently to induce fibrillogenesis and the formation of a gel matrixcontaining cells.

Bell, et al., European Patent Application No. 89309972.1 relates totissue equivalents which have cell types differentiated from progenitorcells without exogenous chemical induction. The tissue equivalent is inthe form of a tissue precusor mixture which is non-gelled, and themixture is injected into the host. The mixture gels and is space fillingupon injection into the appropriate site. The cells must exhibit theability to differentiate without exogenous chemical induction for thetissue equivalent to be effective.

Shing, Y. et al. Cell Biology, Vol. 103, No. 5, Pt. 2 Abstract No. 1107,page 299a (1986) relates to a chondrosarcoma derived growth factor whichis angiogenic in vivo. The chondrosarcoma growth factor is used tostimulate endothelial cell proliferation in vitro.

Bell, et al. Science Vol.211, pp 1052-1054 (1981) relates toskin-equivalent grafts treated with a contractile agent to form acollagen lattice. The lattice is seeded with epidermal cells. Thelattice allegedly permits vascularization of the graft.

Weinberg, C. B., et al., Science Vol. 231: 397-400 (1986), relates to ablood vessel model containing collagen and cultured vascular cells.

Kent, K. C. et al., J. Vascular Surg. Vol. 9, No. 2, pp 271 to 276(1989), relates to endothelial seeding of vascular grafts in dogs, andthe patency of the luminal monolayer. Endothelial cells harvested frombovine aorta, canine external jugular vein and human saphenous vein arecompared.

Hoch, J. et al. J. Surg. Res. vol. 44, No. 5, pp. 545 to 554, relates tothe use of Dacron and polytetrafluoroethylene polymeric grafts, as wellas bovine carotid artery heterografts which were compared in vitro todetermine the extent of endothelial cell adherence.

Noyes, W. F. Proc. Soc. Exp. Biol. Med. Vol. 144, No. 1 pp. 245-248(1973) relates to human liver cell cultures which utilize collagen as asubstrate. Gel-foam sponge is also used as a substrate.

The present invention relates to a transplantable or implantablexenogeneic or allogeneic tissue having immunogenic sites which ifuntreated, would ordinarily induce an immune system response in thepatient, ultimately leading to transplant rejection. Similarly, a methodis described of rendering the transplantable tissue substantiallynon-immunogenic by replacing native cells with allogeneic or autologouscells, reducing the recognition of a transplanted graft as a foreignsubstance without generally suppressing the patient's immune system.

In particular, the present invention relates to transplantable tissuewhich can be treated in accordance with the methods described herein toreduce or prevent untoward immune system reactions which the recipientmay experience in response to the graft, which in turn minimizestransplant rejection. Hence, one object of the present invention is toreduce patient rejection of transplanted tissue.

A further object of the present invention is to increase the supply oftransplantable tissue by treating grafts to render them suitable fortransplant into human patients in need of such treatment.

A further object of the present invention is to facilitate the use ofanimal donors that can supply xenograft donor tissue in virtuallyunlimited quantities. The donor tissue can be transplanted into humanrecipients after the tissue has been treated in accordance with themethods described herein.

Additional objects of the present invention will be apparent to thoseskilled in the art from the teachings herein.

SUMMARY OF THE INVENTION

The invention described herein includes a transplantable bioprostheticgraft tissue which is treated prior to transplant with a growth factorand then exposed to cells which are attracted into the tissue andproliferate in response to the growth factor and populate thetransplantable tissue. Replacement of cells effectively reduces immuneresponses to the tissue, thus improving the effective life of the graftand reducing the frequency, incidence and severity of transplantrejection.

The invention further addresses a method of treating xenogeneictransplantable tissue which comprises exposing the tissue to a growthfactor and then culturing the graft tissue with cells which migrate andproliferate in response to the growth factor, thus populating the tissuewith the cells to enhance the effective life of the tissue upontransplant, and reduce any immunologically mediated adverse effectswhich the graft recipient otherwise experiences in response to thexenogeneic tissue upon transplantion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photomicrograph of control tissue not exposed to growthfactor, and

FIG. 2 is a photomicrograph of tissue exposed to basic fibroblast growthfactor ("bFGF") (2500 ng/ml) and incubated with fibroblasts for 10 days.

DETAILED DESCRIPTION

The terms "tissue", "organ" and "organ part" are used in the generalsense herein to mean any transplantable or implantable tissue, organ ororgan part, the survivability of which is improved by the methodsdescribed herein upon implantation. In particular, the overalldurability and longevity of the implant are improved, and host-immunesystem mediated responses, e.g., graft rejection, are reduced inseverity as well as in frequency, and may be eliminated altogether.

The terms "transplant" and "implant" are used interchangably to refer totissue or cells (xenogeneic or allogeneic) which may be introduced intothe body of a patient to replace or supplement the structure or functionof the endogenous tissue.

The term "autologous" refers to tissue or cells which originate with orare derived from the recipient, whereas the terms "allogeneic" and"allograft" refer to cells and tissue which originate with or arederived from a donor of the same species as the recipient. The terms"xenogeneic" and "xenograft" refer to cells or tissue which originateswith or is derived from a specie other than that of the recipient.

The invention described herein is particularly useful for bioprostheticxenografts in which the major structural component is connective tissuematrix. Examples of such grafts include bioprosthetic heart valves,blood vessels, ligaments and tendons.

Hence, a preferred aspect of the invention encompasses a xenografttreated with a growth factor and incubated with cells that migrate andproliferate in response to the growth factor, thus populating thexenograft, said replacement of cells being effective for reducingallergic complications upon transplant when compared to untreatedxenografts.

Upon treatment of the xenograft with growth factor according to themethods described herein, and upon population of the xenograft withallogeneic or autogenous cells that improve the viability of thexenograft after transplant and reduce any immune response to thexenograft, there is a reduced tendency for thromboemboli to occur,particularly when compared to mechanical heart valves. This results inincreased implant longevity, decreased or slowed degeneration of theimplant, and decreased adverse immune reactions which otherwise mayresult in host rejection.

The preferred growth factor for use herein is fibroblast growth factor,in particular, basic fibroblast growth factor ("bFGF"). When used totreat xenograft implants, such as heart valves, the graft may beinitially exposed to a buffered nutrient medium, and then immersed in asolution containing bFGF. Optionally the graft may be sterilized andrendered acellular using an effective dose of radiation or a cytotoxicsolution prior to treatment with bFGF.

The concentration of growth factor used to treat the xenograft typicallyranges from about 100 ng/ml to 10 mg/ml with a growth factorconcentration for bFGF of about 2.5 mcg/ml being most preferred.

The graft is bathed in the solution containing growth factor for a timeperiod which is effective for causing cells which migrate andproliferate in response to the growth factor to adhere to and penetratethe surface of the xenograft. This, in effect, causes the cells topopulate the xenograft.

To populate (or repopulate) the graft with cells, the graft may bewashed, immersed in a growth factor containing solution, and then placedinto a suitable buffered medium containing the cells which migrate andproliferate in response to the growth factor, thus populating the grafttissue with cells. The graft and cells are cultured together at atemperature and for a time period which are effective for causing thecells to populate and adhere to the graft.

Culture times range from about 3 to 21 days. Culture times may bereduced somewhat by increasing the initial concentration of cells.

When fibroblasts are used as the graft-populating cells, the graft maytypically be immersed in Dulbecco's Modified Eagle medium with 5% serum.The graft is cultured with a primary fibroblast culture for about threedays. Additionally the graft may be secured on the culture plate andincubated at about 37° C. in a humidified atmosphere, until the grafthas been populated with fibroblasts, e.g. 5% CO₂ /95% air. Incubation isconsidered complete when the fibroblasts have populated the graft insuch a manner that the graft appears histologically similar to a freshgraft. (e.g., a normal cell distribution).

Essentially any buffered physiological salt solution containing proteincarriers can be employed.

Preferred buffers for use with the growth factor include sodiumphosphate/glycerin/bovine serum albumin ("BSA"). These buffers typicallyare used to provide a physiologically acceptable pH, such as about 7.0to 7.6.

The cells which are used to populate the graft can be varied within widelimits, and different types of cells can be used in differentcircumstances, depending upon the site and size of the transplant, thenature of the tissue to be replaced, the allergic sensitivity (orhypersensitivity) of the patient and other factors.

The graft may be sterilized prior to treatment with the growth factor,or treated to kill off the endogenous cells in the graft prior totreatment with growth factor and subsequent graft population. This mayreduce the likelihood of microorganismal contamination as well as theimmunogenicity of the graft prior to graft population and implantation.A preferred method for sterilizing grafts prior to population utilizesradiation exposure, e.g., x-rays in lethally effective doses.Alternatively, antibiotics, antibacterials and cytotoxic agents innormally effective doses may be used.

A preferred aspect of the invention involves the use of autogenous cellsin the process described herein. In this instance, a tissue sample istaken from the patient prior to transplant surgery. The tissue istreated in accordance with the methods described herein to producefibroblasts or other cells which are then used to repopulate the graft.By immersing the graft in growth factor and a culture of autogenouscells, and by populating the graft with cells derived from the resectedtissue taken from the patient, an adverse immune system response andultimately graft rejection can be minimized or avoided.

The cell source tissue can be selected to match the tissue to betransplanted. For example, if a blood vessel is to be transplanted,cells can be taken from a patient, healthy blood vessel and used as thesource of cells for graft population. In this fashion, the healthy graftcan be very closely matched to the patient's diseased tissue.

This aspect of the invention is particularly useful when the transplantpatient is highly allergic, or if the tissue is highly immunogenic, suchas with respect to transplantable blood vessels.

Alternatively, cell lines can be used to repopulate the graft which aresubstantially non-immunogenic. Cells which elicit no more than a weakallergic response are used to populate the graft prior to transplant.

Method for Isolation of Fibroblasts

The tissue, for example skin or heart valve leaflet, is cut into 1 mm³pieces using a sterile dissection technique. Groups of 10 pieces arethen placed in 35 cm² tissue culture dishes with approximately 1 ml ofculture medium (DMEM+10% FCS). It is important that the pieces of tissueremain attached to the plastic surface of the culture dish; if thetissue detaches, the amount of culture medium should be reduced.Incubate for 1 week at 37° C. in a humidified culture incubator. After 1week of incubation, each piece of tissue is surrounded by a denseoutgrowth of fibroblasts. Epithelial cells may also be present but arelost during subsequent cell culturing. The fibroblasts are removed witha plastic scraper or by collagenase digestion after rinsing the cellswith a calcium and magnesium-free buffered salt solution, and placed inlarger cell culture vessels with fresh culture medium. The cell culturescan be expanded in this manner. The contents of one flask can be dividedand placed into three larger vessels, and this process can be repeatedabout once a week for at least 10 weeks. These flasks of fibroblasts arethen utilized as a cell source. Cells obtained in this manner arepreferable to commercially available cell lines, because most cell linesare genetically modified and are no longer responsive in a normal mannerto growth regulators (such as FGF).

The fibroblasts can be either immunologically matched allogeneic cells,such that the recipient does not recognize them as foreign, orautologous cells, in which case the donor and recipient are the sameindividual.

Results

A study was performed using canine leaflets and bovine fibroblasts.Mitral valve leaflets were aseptically harvested from a dog cadavershortly after it was killed. The leaflets were divided into sections andplaced in petri dishes containing 5 ml NaH₂ PO₄ /glycerin/BSA buffer.The leaflets were irradiated with 4,000 cGy of 6 MV x-rays to kill thedonor cells. The leaflets were then placed in HBSS (Hanks Balance SaltSolution) with 0.25% trypsin for 10 minutes to remove any residualendothelial cells. The trypsin was inactivated by adding cold culturemedium with 5% serum. The leaflets were washed and placed in NaH₂ PO₄/glycerin/BSA buffer. Human recombinant bFGF was added to the NaH₂ PO₄/glycerin/BSA buffer in the following concentrations: 0, 50, 500, and2,500 ng/ml and incubated for 4.5 hours.

The stock solution of bFGF was prepared with sodium heparin added in a3:1 bFGF:heparin (w/w) ratio. Aliquots and the bFGF stock solution werestored at -70° C.

After incubation, the leaflets were washed in phosphate buffered salineand placed in DMEM (with non-essential amino acids andpenicillin/streptomycin) with 0.5% fetal calf serum ("FCS"). Bovinefibroblasts, which had previously been obtained from calf aorta bystandard explant techniques were added to the heart valve leaflets at2×10⁴ cells/ml. The heart valve leaflets were then secured to the bottomof the plate with small weights and incubated for 10 days at 37° C. in ahumidified 5% CO₂ and 95% air environment.

Following incubation, valve sections were placed in formalin forhistopathological analysis. The analysis demonstrated that there was abFGF dose-dependent increase in fibroblast ingrowth into the heart valveleaflets. For example representative micrographs in FIG. 1 show thecontrol leaflets not exposed to bFGF were essentially acellular, whereasleaflets exposed to 2500 ng/ml bFGF shown in FIG. 2 were well populatedwith cells. These results demonstrate that fibroblasts will populate anirradiated FGF-treated xenograft.

The description contained herein contains the preferred embodiments ofthe invention. However, numerous alternative embodiments arecontemplated as falling within the scope of the invention.

I claim:
 1. A method of reducing the immunogenicity and improving thelongevity of an implantable natural tissue comprising:harvesting abodily tissue suitable for transplantation into a patient, treating thebodily tissue prior to implantation with a growth factor, and populatingthe tissue throughout with cells that respond to the growth factor priorto implantation such that the bodily tissue retains the naturalmorphology of untreated tissue, wherein the cells are effective forreducing the patient's immune response to the tissue upon implant.
 2. Amethod of reducing transplant tissue rejection comprising:harvesting abodily tissue suitable for transplantation into a patient, treating thebodily tissue prior to implantation with a growth factor, and populatingthe treated tissue with cells that respond to the growth factor prior toimplantation such that the bodily tissue retains the natural morphologyof untreated tissue, wherein the cells are effective to reduce tissuerejection upon transplant.
 3. The method of claim 1 wherein the tissueis sterilized prior to the treatment with growth factor.
 4. The methodof claim 2 wherein the tissue is sterilized prior to treatment with thegrowth factor.
 5. The method of claim 4 wherein the tissue is exposed toradiation prior to treatment with the growth factor.
 6. The method ofclaim 3 wherein the tissue is exposed to radiation prior to treatmentwith the growth factor.
 7. A method according to claim 1 wherein thecells comprise fibroblast cells.
 8. A method according to claim 1wherein the growth factor is effective on fibroblast cells.
 9. A methodaccording to claim 8 wherein the growth factor effective on fibroblastcells is basic fibroblast growth factor.
 10. A method according to claim8 wherein the growth factor effective on fibroblast cells is acidicfibroblast growth factor.
 11. A method according to claim 2 wherein thecells comprise fibroblast cells.
 12. A method according to claim 2wherein the growth factor is effective on fibroblast cells.
 13. A methodaccording to claim 12 wherein the growth factor effective on fibroblastcells is basic fibroblast growth factor.
 14. A method according to claim12 wherein the growth factor effective on fibroblast cells is acidicfibroblast growth factor.
 15. A method of reducing immunogenicity andimproving the longevity of an implantable natural tissue, comprisinga)harvesting a bodily tissue suitable for transplantation into a patient,b) treating the bodily tissue to eliminate endogenous cells from thetissue; and c) repopulating the bodily tissue with cells in the presenceof growth factor to promote cell-repopulation of the implantable tissue,wherein the bodily tissue retains the natural morphology of untreatedtissue and the cells respond to the growth factor and are effective toreduce tissue rejection upon transplant.
 16. The method of claim 15wherein the tissue is selected from the group consisting of connectivetissue, heart valves, blood vessels, ligaments and tendons.